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Human Molecular Genetics Advance Access originally published online on August 2, 2006
Human Molecular Genetics 2006 15(18):2659-2672; doi:10.1093/hmg/ddl194
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Müller glia cells

Agnes G.S.H. van Rossum1, Wendy M. Aartsen1, Jan Meuleman1, Jan Klooster2, Anna Malysheva1, Inge Versteeg1, Jean-Pierre Arsanto3, André Le Bivic3 and Jan Wijnholds1,*

1 Department of Neuromedical Genetics, 2 Department of Retinal Signal Processing, The Netherlands Institute for Neuroscience (NIN), Royal Netherlands Academy of Arts and Sciences (KNAW), Meibergdreef 47, 1105 BA Amsterdam, The Netherlands and 3 UMR CNRS 6216, Institute of Developmental Biology of Marseille-Luminy, Faculté des Sciences de Luminy, Case 907, 13288 Marseille Cedex 09, France

* To whom correspondence should be addressed. Tel: +31 205664597; Fax: +31 205666121; Email: j.wijnholds{at}nin.knaw.nl

Received April 26, 2006; Accepted July 26, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal diseases including Leber's congenital amaurosis (LCA) and retinitis pigmentosa type 12. The CRB1 transmembrane protein localizes at a subapical region (SAR) above intercellular adherens junctions between photoreceptor and Müller glia (MG) cells. We demonstrate that the Crb1–/– phenotype, as shown in Crb1–/– mice, is accelerated and intensified in primary retina cultures. Immuno-electron microscopy showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. Human CRB1, introduced in MG cells in Crb1–/– primary retinas, was targeted to the SAR. RNA interference-induced silencing of the Crb1-interacting-protein Pals1 (protein associated with Lin7; Mpp5) in MG cells resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and partial loss of Crb3. We conclude that Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Polarity of neuronal cells and the formation of junctions between cells that build up the complex network of the retina are essential for retina integrity (1). Cellular junctions contribute to the stability and communication between retinal cells (2). At the outer limiting membrane (OLM), photoreceptor cells (PRCs) are connected to Müller glia (MG) cells via adherens junctions (AJs). Tight junctions (TJs) are not present at the OLM. Immediately above the AJs, a protein complex is located at the so-called subapical region (SAR) (3). At the SAR, Crumbs family members (Crb1, Crb2 and Crb3) are found in complex with Pals1 (proteins associated with Lin seven 1), also known as Mpp5 (4–6). In this complex, Pals1 functions as an adaptor protein. Its PSD-95/Dlg/ZO-1 (PDZ) domain can bind to the cytoplasmic ERLI motif of Crb1 and Crb3, and its L27 domain can bind to the L27 domain of Patj (Pals1 associated TJ protein) and Mupp1 (multiple PDZ domain protein 1; Mpdz) (5–8). The Crb/Pals1/Patj complex is connected to the Par6/Par3/aPKC complex, containing Par6 and Par3 (partition defective proteins) and aPKC (atypical protein kinase C). The Par3/Par6/aPKC complex is involved in the regulation and maintenance of cellular polarity and AJs in several cell types (9–11) and can bind directly to Crb3 (12). Furthermore, aPKC{lambda} is required in differentiating photoreceptors for proper lamination of the mouse retina (13), and the zebrafish Par3 ortholog (Pard3) is required for separation of the eye fields and retinal lamination (14).

In Drosophila, mutations in each member of the Crumbs–Stardust–DPATJ complex (homologues of Crb1/Crb2/Crb3, Pals1 and Patj, respectively), resulted in epithelial polarity or retinal morphogenesis defects (15–19). The Crb–Pals1–Patj complex position at the SAR in mice is in part comparable to its position at the stalk membrane just apical to the AJs in the Drosophila compound eye. Mutations in both Stardust and Crumbs gave rise to the same rough eye phenotype, which includes shortened stalk membranes and distorted rhabdomeres. Mutations in DPATJ demonstrated DPATJ to be necessary for the stability of the Crb complex and crucial for stalk membrane development and rhabdomere maintenance during late pupal stages (20,21). This proves the Crumbs–Stardust–DPATJ complex to be indispensable for Drosophila eye integrity (15,22–24).

In humans, mutations in the CRB1 gene lead to a variety of retinal degenerative diseases such as Leber's congenital amaurosis (LCA), retinitis pigmentosa type 12 (RP), retinitis pigmentosa with coats-like exudative vasculopathy and pigmented paravenous chorioretinal atrophy (25–28). These human retinal degenerative diseases can be mimicked in part by the Crb1rd8 and Crb1–/– mouse models (3,29). Loss of photoreceptors and structural integrity of the OLM is observed in both mutant models and appears in a focal manner adjacent to apparently normal regions. The retinal disorganization is due to the loss of maintenance of adhesion between photoreceptors and MG cells (3,29). This phenotype can be visualized as irregular white spots on fundus pictures and is most striking in older animals.

In mammalian epithelial cells, loss of PALS1 or replacing its PDZ domain caused disturbed TJ formation and polarity defects (30,31). This suggests an important role for the adaptor protein PALS1 in protein complex formation in cellular junctions. In addition, mutations of the zebrafish gene nagie oko (nok, homologue of PALS1) showed nok to be essential in cellular patterning of the retina, suggesting PALS1 to be indispensable for zebrafish eye integrity (32,33).

Cell polarity or retinal defects caused by loss of CRB1 or PALS1 are shown in different organisms or cell systems. In this study, we investigated the significance of the Crb–Pals1–Patj complex in the mammalian retina. We performed gain- and loss-of-function studies involving Crb1 and Pals1 in wild-type (WT) and Crb1–/– primary retina cultures using an in vitro electroporation method. We found, by using immuno-electron microscopy (EM), that at the SAR Crb1 was mainly detectable in MG cells but barely in PRCs, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. RNA interference (RNAi)-induced silencing of Pals1 in MG cells resulted in a loss of Crb1, Crb2, Mupp1 and Veli3 (mLin7C, Mals-3) at the SAR, and partial loss of Crb3, but not of Patj, Par3, Mpp4, ß-catenin or N-cadherin. Our results demonstrate that the scaffold protein Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Development of Crb1–/– retina in vitro: acceleration of the Crb1–/– phenotype
In previous studies, we generated and characterized Crb1–/– knockout mice (3). In retinas from 3 to 6 months old Crb1–/– mice, double PRC layers or half-rosettes manifested and these morphological changes together with expanded retinal degeneration augmented with age and light exposure (3). Retinal disorganization manifested in less than 20% of a specific region of the retina, the inferior temporal quadrant. In addition to these in vivo studies, here we used an in vitro primary retinal organ culture method (34,35) to investigate the development and structural integrity of Crb1–/– retinas. Retinas of 1.5 days old mice were isolated and maintained in culture up to 28 days in vitro (DIV). Although the organ-cultured retina is thinner than the retina of an age-matched in vivo control eye (Fig. 1N versus O) (35), the retinal explants of WT retinas isolated without RPE developed normally with all retinal layers formed but with underdeveloped photoreceptor outer segments. The layers remained normal up to at least 4 weeks of culturing (Fig. 1 N) (data not shown). During the first 6 days in organ culture, retinas from Crb1–/– mice cannot be distinguished from WT retinas. Interestingly, after 6 DIV, morphological alterations showed up in the Crb1–/– retina appearing as white spots (Fig. 1C–F, defined by the black line). Those white spots extend to 30 up to 80% of the retina at 14 DIV and represent retinal folds and photoreceptor half-rosettes in the outer nuclear layer (ONL) (Fig. 1J–M). In WT retinas, some white spots appeared as well, but were usually confined to the edges of the organ culture, covered less than 10% of the retina area and is normal for WT retina cultures as shown previously (35). Thus, the Crb1–/– morphogenetic phenotype, as seen in Crb1–/– mice at 3–6 months of age, is significantly accelerated in primary retina cultures and emerges after 6 DIV.


Figure 1941
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  		Figure 1. Disturbed development of Crb1–/– retina in vitro. Pictures of mouse retinal explants of Crb1–/– (A–H) and WT retinas (I) grown in organ culture were taken with an inverted microscope at different DIV. Note the appearance of morphological alterations (defined by the black line and closed circles), appearing as white spots in the Crb1–/– retina (arrowhead). Those white spots develop after 6 DIV and cover 30–80% of the retina at 14 DIV (n=14) and later on, and represent retinal folds and half-rosettes in the ONL. The retina is cultured with its photoreceptors facing the membrane as the undermost layer, whereas the GCL is the uppermost layer. The asterisk represents the position of the optic nerve, black line and closed circles define morphological alterations in the Crb1–/– retina and the black arrows represent the scissor cuts that allow the retina to flatten. Although RPE sheets are detached during the isolation process, a few RPE cells always remain, seen as black dots (I). Scale bar in (I): 100 µm. (A–F) were taken with the same magnification as in (I). Scale bar in (G and H): 250 µm. Light microscopic pictures of paraffin sections stained with haematoxylin from Crb1–/– (J–M) and WT (N) retinal explants were taken after various DIV as well as pictures of retinas from WT (O) and Crb1–/– mouse (P) at 14 days postnatal (P14) for comparison. The thickness of the retinal layers depends on the distance to the optic nerve, being thickest closest to the optic nerve. (J) During paraffin embedding, the retina folded double over: the arrows represent the layers from GCL to IS. (N) Representative picture of WT retina from 13 to 27 DIV. The stratification of the tissue is well preserved, including the delicate structure of the inner segments. (P) Note the wavy OLM and PRC nuclei above the OLM in the Crb1–/– P14 retina, but no (half-)rosette formation. RPE, retinal pigment epithelium; OPL, outer plexiform layer. (J–P) were taken with the same magnification as in (N). Scale bar: 100 µm.

 
Interruptions at the OLM and disturbances of MG cells in Crb1–/– retina explants
In the retina, the Crb1 protein localizes at the SAR of the OLM, just above the intercellular AJs between photoreceptor and MG cells (3). We examined WT retinal explants of 27 DIV for their localization of proteins in and around the OLM (Fig. 2) (data not shown). Members of the Crumbs family (Crb1, Crb2, Crb3), as well as the multiple PDZ domain proteins Mupp1 and Patj, the MAGUK proteins Mpp4 and Pals1, the polarity proteins Par3 and aPKC, and proteins connected to the AJs, e.g. ZO-1, ß-catenin, {delta}-catenin, p120 catenin and N-cadherin, are all detectable at the OLM. Also proteins expressed in MG cells (glutamate synthetase), microvilli of the MG cells (CD44) and proteins of the inner segments (F-actin, moesin) are properly localized and the underdeveloped photoreceptor outer segments do express rhodopsin. Cone photoreceptor outer segments could also be detected by peanut agglutinin suggesting normal numbers of cones (data not shown). From these results, we conclude that the OLM and inner segments of cultured WT retinas are properly developed.


Figure 1942
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  		Figure 2. Proper localization of OLM proteins in WT retina explants. Confocal images of several OLM proteins of WT retina explants of 27 DIV. The stratification of the tissue is well preserved, including the delicate structure of the inner segments. However, the formation of the outer segments is affected due to the absence of an RPE sheet (54). (A) As a marker for AJs, ß-catenin localized more basal to the SAR protein Pals1. (B) F-actin localizes at the inner segments above Pals1 staining at the SAR. (C) Crb1 localizes, like Pals1, at the SAR and moesin localizes, like F-actin, at the inner segments. (D) Rhodopsin, a marker for outer segments, is expressed above the inner segments that were marked by F-actin staining. (E) Weak Par3 staining is observed at the OLM. IS, inner segment. Scale bar: 10 µm.

 
Immunohistochemical detection of the above-mentioned proteins on Crb1–/– retina explants of 27 DIV revealed (i) a disturbed ONL with retinal folds, half-rosettes and (ii) interruptions of the OLM, not within, but adjacent to the half-rosettes. Interestingly, OLM proteins (of the SAR and AJ), inner and outer segment markers, and MG cell markers are properly localized within the half-rosettes but not in the adjacent regions (Fig. 3A and D, moesin and Mupp1, respectively) (data not shown). The regular pattern of MG cells as seen in WT retinas is grossly disrupted in Crb1–/– retinas, particularly in areas of clusters of displaced photoreceptors adjacent to half-rosettes (Fig. 3B). Damage to the retina is detected in MG cells at an early stage by increased expression and/or changed subcellular localization of the intermediate filament protein glial fibrillary acidic protein (GFAP) (36). In Crb1–/– retina explants, like in Crb1–/– mutant mice (3), GFAP immunoreactivity is increased in areas where the OLM is interrupted (Fig. 3B–D) compared with WT retina explants (Fig. 3E). In addition, the microvilli of the MG cells are disturbed in these areas as detected by CD44 staining, but not within half-rosettes themselves (Fig. 3C). Taken together, the Crb1–/– phenotype, which is characterized by OLM interruptions, disturbances of MG cells and formation of retinal folds and half-rosettes, is significantly accelerated and intensified in primary retina cultures.


Figure 1943
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  		Figure 3. Interruptions of the OLM and disturbances of MG cells in Crb1–/– retina explants. Fluorescent microscopic (A and B) and confocal images (C–E) of WT and Crb1–/– retinas of 27 DIV. (A) Crb1 localizes at the SAR and moesin at the inner segments. Note that in half-rosettes (arrow), proper localization of OLM proteins demonstrates an intact OLM. In contrast, their staining pattern is disturbed in affected areas, adjacent to the half-rosettes. Nuclei are stained with Hoechst (blue). (B) In WT retinas, glutamate synthetase immunoreactivity is restricted to MG cells. In Crb1–/– retinas, the regular array of MG cells is grossly disrupted in areas of clusters of displaced photoreceptors adjacent to half-rosettes (arrow) and exactly at that location where GFAP immunoreactivity at the level of the OLM is increased. (C) CD44 is a marker for the microvilli of the MG cells which is increased in disturbed areas in Crb1–/– retinas where the OLM is interrupted but not in WT retinas (E). In contrast, CD44 staining is properly localized within the half-rosettes (arrow). (D and E) In WT retina, Mupp1 localizes at the SAR. As a marker for an intact OLM (arrowhead in Crb1–/– retina), Mupp1 localizes properly at the SAR within but not adjacent to half-rosettes (arrow). Normal GFAP staining in WT retina, but increased GFAP immunoreactivity in Crb1–/– retina adjacent to half-rosettes. Scale bar in (A and B): 100 µm. Scale bar in (C–E): 10 µm.

 
At the SAR, Crb1 locates primarily in MG
Pre-embedding electron microscopic immunohistochemistry (37) on WT eyes of 3-month-old mice revealed strong Crb1 immunoreactivity at the SAR in microvilli of polarized MG cells, but barely detectable signals in PRCs (Fig. 4B–D). Crb1 immunoreactivity was absent in Crb1–/– retinas (Fig. 4A). Pals1 immunoreactivity was detectable at the SAR in both MG and PRCs in WT as well as in Crb1–/– eyes (Fig. 5), which is consistent with immunofluorescent staining of Pals1 on Crb1–/– eyes (3) and Crb1–/– primary retinas (discussed earlier). Post-embedding electron microscopic immunohistochemistry (38) on adult WT eyes revealed immunoreactivity of Crb2 (Fig. 6) and Crb3 (Fig. 7) at the SAR in both MG cells and photoreceptor inner segments. In addition, also Mupp1 (Supplementary Material, Fig. S1) and Patj (Supplementary Material, Fig. S2) were found at the SAR in MG and photoreceptor inner segments. These results demonstrate that MG cells do express Crb family proteins and interactors endogenously.


Figure 1944
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  		Figure 4. Endogenous Crb1 locates at the SAR in MG cells. Immuno-electron micrograph from retinas of Crb1–/– (A) (n=2) and WT (B–D) (n=2) eyes of 3-month-old mice immunolabelled for Crb1. (A) No Crb1 protein was detected in the MG or PRCs at the SAR of Crb1–/– mice. (B–D) The Crb1 protein is primarily expressed at the SAR in the microvilli of the MG cells (arrows) just above the AJ and barely in photoreceptor IS. Notice that Crb1 immunolabelling in the WT retina is not restricted to the plasma membrane. PR, photoreceptor; IS, inner segment; v, microvilli; N, nucleus. Scale bar in (A and B): 1000 nm. Scale bar in (C and D): 500 nm.

 


Figure 1945
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  		Figure 5. Endogenous Pals1 locates at the SAR in MG cells and PRCs. Immuno-electron micrograph from retinas of WT (A) (n=2) and Crb1–/– (B) (n=2) eyes of 3-month-old mice were immunolabelled for Pals1. Pals1 protein was detected in both MG and PRCs at the SAR of WT (A) and Crb1–/– mice (B). PR, photoreceptor; IS, inner segment; v, microvilli. Scale bar: 500 nm.

 


Figure 1946
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  		Figure 6. Endogenous Crb2 locates at the SAR in MG cells and PRCs. (A) Electron micrograph from adult WT eyes showing morphological features to more clearly identify the sites of Crb2 immunolabelling in (B). Photoreceptor IS displaying many cytoplasmic structures, especially a very large Golgi apparatus, are observed around an apical MG cell. (B) Immuno-electron micrograph showing Crb2 labelling in a part of the OLM similar to that seen in (A) (bottom of the picture). Clusters of gold particles are found close to the plasma membrane, both in the IS and in the adjacent MG cell where labelling (arrowheads) is seen near the cytoplasmic electron-dense plaque of one of the two AJs. IS, inner segment; v, microvilli; G, Golgi apparatus. Scale bar in (A): 200 nm. Scale bar in (B): 100 nm.

 


Figure 1947
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  		Figure 7. Endogenous Crb3 locates at the SAR in MG cells and PRCs. (A and B) Electron micrograph from adult WT eyes showing morphological features to more clearly identify the sites of Crb3 immunolabelling in structures observed in (C–E). Transverse sections through the OLM. The electron-dense plaque material on both plasma membranes indicates the AJs, which connect the photoreceptor inner segments with the interspersed MG cells. Note that the cytoplasm of photoreceptor IS is rich in organelles, including a large Golgi apparatus, whereas the MG cells seen here in their most apical part with microvilli processes mainly contain cytoskeletal fibrillar material. (C) Clusters of immunogold particles indicate the presence of Crb3 on each side of the AJ (arrows), i.e. near both plasma membranes of photoreceptor IS and MG cell. (D) Immuno-electron micrograph showing Crb3 labelling (arrows) in the vicinity of the plasma membranes of the two types of cells. (E) A higher magnification, showing Crb3 staining close to the plasma membrane both in the IS and in MG apical microvilli. IS, inner segment; v, microvilli; G, Golgi apparatus. Scale bar in (A): 200 nm. Scale bar in (B–E): 100 nm.

 
To reconstitute Crb1 in primary Crb1-deficient retinas, we used an in vitro electroporation technique (39). CMV-promotor-driven human CRB1 (hCRB1) was electroporated in 1.5-day-old Crb1–/– mouse retina and analysed at 17 DIV. The electroporation efficiency was relatively low, but sparsely, clusters of on average five spots of hCRB1 were detected at the SAR just above the ß-catenin staining, which is a marker for AJs (Fig. 8A). Double labelling with anti-Crb1 and anti-glutamate synthetase (an MG cell marker) revealed that hCRB1 immunoreactivity was detected at the SAR in individually transfected MG cells (Fig. 8B), showing that CMV-driven CRB1 is expressed and CRB1 properly localized in MG cells.


Figure 1948
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  		Figure 8. Exogenous electroporated hCRB1 is directed to the SAR in MG cells. Confocal images of Crb1–/– retina of 17 DIV electroporated in vitro with AAV2-CMV-driven human CRB1 plasmid. (A) Introduction of CRB1 resulted in human CRB1 localization at the SAR above ß-catenin staining, which is a marker for AJs. (B) Double labelling of Crb1 with MG cell marker glutamate synthetase. Note that exogenous introduced human CRB1 localizes at the SAR above individual MG cells (arrows). Scale bar: 10 µm.

 
Downregulation of Pals1 in MG cells results in reduction of Crb family members and its interactors from the SAR
Silencing of Pals1 in MDCKII cells leads to TJ and polarity defects (30,31) and mutations in the Pals1 homologue in zebrafish (nagie oko; nok) cause defects in the retinal patterning mechanism (32). To investigate the consequences of Pals1 silencing in the mouse retina, we used short hairpin RNAs (shRNAs) (40) and an in vitro electroporation technique (39). Initially, five pSUPER-based shPals1 constructs were generated; one shRNA construct, corresponding to nucleotides 1970–1988 of the GUK domain of mouse Pals1 (3-shPals1), revealed specific and efficient silencing of a GFP-tagged Pals1 oligo (Supplementary Material, Fig. S3A).

The pSUPER-based 3-shPals1 construct was subcloned into an AAV2-vector containing a CMV-GFP expression marker (CMV-GFP–shPals1). Retinas of P1.5-old mice were electroporated in vitro with the CMV-GFP–shPals1 construct or control CMV-GFP vector and analysed at 17 DIV. Approximately 20–40% of the surface area of the retina contained visible GFP patches. For both constructs, GFP is detected in MG cells and sporadically in PRCs (Fig. 9A). Immunostaining with anti-Pals1 and anti-GFP showed that Pals1 immunoreactivity was silenced in green cells containing the CMV-GFP–shPals1 vector (Fig. 9B and C).


Figure 1949
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  		Figure 9. Silencing of Pals1, but not Mpp4, at the SAR in MG cells. (A) Fluorescent microscopic images of WT retina electroporated with AAV2-CMV-GFP. Nuclei were stained with Hoechst (blue) and the electroporated MG cell (arrow) emitted the GFP fluorescence. (B and C) Fluorescent microscopic images of WT retina electroporated with AAV2-CMV-GFP–shPals1, double labelled with anti-Pals1 [white in (B) and red in (C)] and anti-GFP (green). Pals1 immunoreactivity was suppressed in the GFP-positive MG cells (arrows). PRCs were sporadically GFP positive (arrowheads). Nuclei were stained with Hoechst (blue). (D) Confocal images of WT retina electroporated with AAV2–CMV-GFP–shMpp4, double labelled with anti-GFP (green) and anti-Mpp4 (red). Note that Mpp4, which is expressed in PRCs but not in MG cells (41), is not reduced at the OLM. The AK4 antibody directed against Mpp4 shows some background staining in the retina as detected in Mpp4–/– mice (47). OPL, outer plexiform layer; IPL, inner plexiform layer. Scale bar: 10 µm.

 
As a control, we used a similar approach for Mpp4, which is expressed in PRCs, but not in MG cells (41). Four pSUPER-based shMpp4 constructs were created, and one construct (5-shMpp4), corresponding to nucleotides 1726–1744 of the GUK domain of mouse Mpp4, silenced most efficiently the GFP-tagged Mpp4 gene (Supplementary Material, Fig. S3B). Again, in vitro electroporation of AAV2-vector containing CMV-GFP–shMpp4 resulted predominantly in GFP-positive MG cells. Immunostaining with anti-Mpp4 and anti-GFP did not reveal downregulation of Mpp4 at the SAR (Fig. 9D), which is expected, because Mpp4 is not expressed in MG cells. These results demonstrate that Pals1, but not Mpp4, is expressed in the MG cells and that endogenous Pals1 can specifically be silenced.

Next, we examined the effect of Pals1 suppression on the expression and/or localization of OLM proteins. Double labelling showed that silencing of Pals1 expression in MG cells resulted in loss of localization of Crb1 and Crb2 proteins and partial loss of Crb3 (Fig. 10A–C). Reduced levels of Mupp1 and Veli3 immunofluorescence were also observed (Fig. 10E and F). The expression of Patj, Mpp4, Par3 (SAR proteins), ß-catenin, N-cadherin, ZO-1 (AJ proteins) and glutamate synthetase (MG cell marker) were unchanged by Pals1 suppression (Fig. 10D) (data not shown). Taken together, we demonstrate that all Crumbs family members (Crb1, Crb2 and partially Crb3), Mupp1, Pals1 and Veli3 are expressed in MG cells and that the expression and/or localization of Crb1, Crb2, Crb3, Mupp1 and Veli3 at the SAR are dependent on Pals1 expression.


Figure 19410
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  		Figure 10. Silencing of Pals1 in MG cells resulted in loss of localization of Crb family members and its interactors from the SAR. Confocal images of WT retina electroporated with AAV2–CMV-GFP–shPals1. Triple labelling of anti-GFP (green), anti-Pals1 (red) with anti-Crb1 (blue) is shown in (A), anti-Crb2 (blue) in (B) and anti-Crb3 (blue) in (C). Delocalization of Crb3 is partial as marked by asterisks. Double labelling of anti-Pals1 (red) with anti-glutamate synthetase (green) in (D) demonstrates no delocalization of glutamate synthetase. Double labelling of anti-GFP (green) with anti-Mupp1 (red) (E) and anti-Veli3 (red) (F). Pals1 immunoreactivity is suppressed in the GFP-positive MG cells, and as a result, Mupp1 and Veli3 are delocalized (arrows). Thus, Crb1, Crb2 and Crb3, Mupp1 and Veli3 (arrows), but not glutamate synthetase, are delocalized upon silencing of Pals1. Scale bar: 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
In this study, we show that loss of function of members of the Crb complex in mammalian retina can bring new insights into the organization of this complex, which has important implications for understanding the role of CRB1 mutations in human retinal diseases. We demonstrate that Pals1 expression in MG cells is required for correct localization of Crb1, Crb2, Crb3, Mupp1 and Veli3, because RNAi-induced silencing of Pals1 in MG cells from WT primary retinas resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and a partial loss of localization of Crb3 at the SAR. Immuno-EM revealed that at the SAR Crb1 was mainly localized in microvilli of MG cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both MG and PRCs. Electroporation of CMV-driven human CRB1 in MG cells in Crb1–/– mouse retinas demonstrated that hCRB1 was properly targeted to the SAR.

In this paper, we show with immuno-EM that Crb1 immunoreactivity is strong at the SAR in MG cells, but barely detectable in PRCs. In Drosophila, Crumbs is localized at the stalk membrane of PRCs, a structure that corresponds with the SAR in mice. Initially, Pellikka et al. (23) showed in mice that Crb1 protein localizes at the OLM in cone and rod photoreceptor inner segments and MG cells. Expression of Crb1 in both cell types was supported by the mRNA in situ hybridization pattern of Crb1 as shown by den Hollander et al. (42), in which the photoreceptor layer and some cells at the inner nuclear layer (INL) expressed Crb1 mRNA. Nuclei of MG cells reside at the INL. For that moment, those results led to the conclusion that in the retina Crb1 is expressed by photoreceptor and MG cells. Immuno-EM experiments revealed now that Crb1 is barely detectable in PRCs. We cannot exclude that the Crb1 mRNA as detected in photoreceptors with an mRNA probe corresponding to the extracellular domain might correspond to splice variants lacking the intracellular domain (29) or the secreted extracellular form of Crb1 (43). As yet, we are not able to detect those Crb1 variants with our antibodies that are directed against the intracellular protein domain only. Immuno-EM experiments of other Crb complex proteins demonstrated that Crb2, Crb3, Patj, Pals1 and Mupp1 localize at the SAR in MG as well as in PRCs. However, Pals1–Crb1, Pals1–Crb2, Pals1–Mupp1 and Pals1–Veli3 complexes might be relatively more present in MG cells than in PRCs, whereas the partial loss of Crb3, after RNAi-induced Pals1 silencing, suggests remaining Crb3 in complexes in PRCs. This study elucidates in part the distribution of expression of Crb complex proteins in MG and PRCs.

PRCs need MG cells for structural and metabolic support and for the establishment and maintenance of apical–basal polarization and cell adhesion. The MG network plays a critical role in neural cell death during retinal degeneration. In light-degenerated retinas, MG cells can invade the photoreceptor layer from the inner part of the retina and increase the production of growth factors to prevent PRC death (44,45). Loss of Crb1 function in MG cells, as caused by mutations, might disturb maintenance of MG-PRC adhesion and retina integrity. In Crb1–/– primary retina cultures, we observed loss of structural integrity of the OLM, leading to displacement of photoreceptors and the formation of retinal folds and half-rosettes. Particularly in areas adjacent to half-rosettes, the OLM is interrupted and the regular pattern of MG cells is grossly disrupted accompanied by increased GFAP and CD44 expression in the MG cells. Disruption or retraction of the apical Müller microvilli visualized by CD44 has also been described as phenotype of the Crbrd8 mouse, together with increased electron density of the MG cell cytoplasma (29). From the Crb1 phenotypes as described by the Crbrd8 (29) and Crb1–/– mice (3), together with our present results on primary Crb1–/– retina cultures, we conclude that lack of Crb1 in MG cells results in loss of adhesion between MG cells and photoreceptors followed by structural alterations of both photoreceptors and MG cells. Changes in length of photoreceptor inner and outer segments as detected in Crbrd8 mice (29), but not in Crb1–/– mice, are therefore indirect effects. In the retinas of Crb1–/– mice, OLM proteins including Crb2 and Crb3 as well as AJ markers were localized as in WT retinas. Delocalization of these proteins is observed in foci of the retina from 3-month-old Crb1–/– mice where MG and PRCs lost their structure (3). In Crb1–/– retina explants, OLM and AJ markers, including Crb2 and Crb3, are localized at the OLM in half-rosettes, but delocalized adjacent to half-rosettes, which indicates that a considerable part of the retina explant exhibits loss of OLM structure while the OLM is intact within the half-rosette. Remarkably, the retinal disorganization phenotype is accelerated and intensified under in vitro conditions, suggesting that at least one additional, and as yet unknown factor, contributes to the Crb1–/– phenotype. This factor might be a protective factor lacking in cultured retinas. Re-introduction of Crb1 into the MG network could be a possible therapeutic target for inhibition of retinal degeneration. In this study, we were able to properly target human CRB1 to the SAR in MG cells of Crb1–/– retinas. As yet, the efficiency of DNA transfer by electroporation of primary retinas is not as optimal to restore the Crb1–/– phenotype.

Our results place the knowledge of Crb1 and its interactions with other proteins in another perspective. First, in vitro experiments showed that Mpp4 can exist in a complex with Crb1 via intervention of Pals1 (41). Although in the retina, Mpp4, Pals1 and Crb1 colocalize at the SAR, it is not very likely that they exist in one complex in vivo, because Mpp4 is expressed exclusively in PRCs and Crb1 mainly in MG cells. In addition, we and Stohr et al. (46) were not able to show an interaction of Pals1 with Mpp4 in immunoprecipitation assays of retinal lysates. Secondly, we recently showed that Mupp1 co-precipitated Crb1, Pals1 and slightly Mpp4 from retinal lysates, but not Patj (3). In vivo direct interactions have been demonstrated for Pals1–Crb1, Pals1–Mupp1, Pals1–Mpp3, Pals1–Veli3 and Mpp4–Veli3, but not for Pals1–Mpp4 and Mpp3–Mpp4, using immunoprecipitations (3,41,46–48). As yet, those results combined with the results of this study lead to the conclusion that the MG cell SAR contains at least Crb1, Crb2, Crb3, Patj, Pals1, Mupp1 and Veli3. The PRC SAR contains at least Crb2, Crb3, Patj, Pals1, Mupp1 and Mpp4. Localization of Patj seems to be independent of Pals1 expression. Thirdly, Veli3 interacts with Mpp4 and Pals1 in retinal lysates (46,47). Immunolocalization of Veli3 was detected at the OLM, the OPL and in cells of the INL, which might represent MG cells. Veli3–Mpp4 complexes are likely to be located mainly at the OPL and possibly at the OLM, whereas Veli3–Pals1 complexes seem to be present at the SAR in MG cells and possibly in PRCs. Fourthly, Pals1 might be able to bind the Crb2-ERLI domain, because (i) Crb1, Crb2 and Crb3 show a high similarity and Pals1 has been shown to bind to Crb1-ERLI and Crb3-ERLI domains (5,6) and (ii) in this paper, we show that loss of Pals1 results in delocalization of all three Crb family members. Fifthly, although the localization of Crb family members is dependent on the expression levels of Pals1, studies on Crb1–/– mice revealed that loss of Crb1 did not affect Pals1 localization at the SAR (3). Thus, Pals1 localization is not dependent on the expression of the transmembrane protein Crb1. Sixthly, in MDCK cells, suppression of PALS1 or replacing its PDZ domain caused disturbed TJ formation and polarity defects and resulted in loss of PATJ but not of CRB3 expression from TJs (30,31). In contrast, in primary retinas, suppression of Pals1 resulted in a reduction of Crb3 expression to some extent, but not of Patj. This discrepancy might be caused by the differences between MDCK cells and retina tissues. (i) The intercellular interactions in MDCK cells are between cells of one type, whereas in retinas, the interactions are between MG and PRCs. (ii) In MDCK cells, the protein complexes are formed in TJs, whereas in retinas the complexes are in the SAR, which is structurally and functionally different from TJs. (iii) In MDCK cells, only Crb3 is expressed, whereas in retina all three Crb family members are expressed, at least in MG cells. (iv) In MDCK cells, localization of PATJ at the TJ is not exclusively dependent on Pals1 (49). (v) PATJ is required for correct localization of Pals1 and Crb3 at the TJ of Caco2 cells (38).

In Drosophila, the Crumbs–Stardust–DPATJ complex (Crb–Pals1–Patj homologue) is indispensable for Drosophila eye integrity (20,22–24). In zebrafish, nagie oko (nok, Pals1 homologue) is essential for maintaining the correct polarity of the retinal neuroepithelial sheet (32,33). In mouse primary retina cultures, Pals1 is required for correct localization of proteins at the SAR in MG cells. As yet, we did not see morphological disturbances in the retina induced by the silencing of Pals1. This might be due to inefficient DNA transfer or the effect of Pals1 silencing has not been analysed at the right time in order to observe morphological alterations. Still, it is likely that retinal and/or polarity defects as caused by loss of CRB1 will also occur in Pals1 conditional knockout retinas because of the concomitant loss of Crb1 localization. Taken together, the adaptor protein Pals1 has an important role in protein complex formation and cell polarity, but its role may vary in different organisms, tissues or cell types or among physiological conditions. We demonstrated that Pals1 is required for correct localization of Crb1, Crb2, Crb3, Mupp1 and Veli3 at the SAR in polarized MG cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 References
 
Animals
Animal care and experiments were performed in accordance with legislation on animal experiments as determined by the Dutch Veterinary Inspection. WT and Crb1–/– mice (3) were used at a 50%:50% cross of C57Bl/6 and 129/Ola.

In vitro electroporation and retinal explant culture
Mouse pups at postnatal day 1.5 (P 1.5) were decapitated using scissors. The head was disinfected with 70% ethanol and moved to a laminar flow hood where sterile procedures were maintained for the remainder of the preparation. Eyes were enucleated and transferred to a 35 mm Petri dish containing Dulbecco's modified Eagle's media (DMEM) (Gibco 31966-021, Invitrogen, Carlsbad, CA, USA) containing 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco 15140-122, Invitrogen). Using fine forceps (nos. 4 and 5), extraneous connective tissue was removed from the globe. The eye was then placed in fresh DMEM or, when the retina was electroporated, in 1x Hanks' balanced salt solution (HBSS, Gibco, 14185-45, Invitrogen). To harvest and isolate neural retina without RPE, the eye was opened along the ora serrata and the cornea, lens and vitreous were removed. The whole retina was then carefully dissected free from the RPE and sclera with as little disruption and manipulation as possible, leaving a few RPE cells behind. To transfer the retina from one place to another, a disposable 1 ml transfer pipette was used with a cut tip to enlarge the opening. Dissected retinas were transferred into a micro-electroporation chamber (Nepagene, Chiba, Japan, model CUY532, 3 mmx10 mmx 5 mm) containing DNA solution at a concentration of 1 µg/µl in HBSS. DNA transfer was performed by electroporations from the scleral side using the pulse generator CUY21 (Nepagene) with five square pulses of 20 V of 50 ms duration with 950 ms intervals. After dissection or after electroporation, the retina was transferred to a Petri dish with DMEM and four radial cuts were made to flatten the tissues. The retina was transferred to a 0.4 µm pore Millicell culture plate insert (30 mm diameter PICMO3050, Millipore, Billerica, MA, USA) and placed in six-well plates containing ~1.5 ml/well of DMEM medium containing 10% FBS, 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco 15140-122, Invitrogen) in the lower compartment. Whole retinas were mounted flat, with the ganglion cell layer (GCL) at the top and the photoreceptor layer at the bottom. Fine tipped forceps were used to tease and flatten the edges of each retina. All excess medium was removed from the membrane leaving only a moist film covering the tissue. Only minimal manipulations were performed because minor disruptions of the tissue during isolation and plating led to significant distortion over time. Cultures were maintained at 100% humidity, 37°C, 5% CO2 and fed every 2–3 days by replacing 0.75 ml media.

Preparation of retinal sections
Electroporated retinas were harvested 17–28 days (Table 1) after electroporation, fixed with 4% paraformaldehyde (PF) in phosphate-buffered saline (PBS) for 30 min at room temperature, placed in 70% ethanol and routinely embedded in paraffin. Transversal sections, including all retinal layers, were cut at 4 µm, stained with haematoxylin and microscopically examined. The thickness of the retinal layers depends on the distance to the optic nerve, being thickest closest to the optic nerve. For cryosections, fixed retinas were washed in PBS, cryoprotected in 15% sucrose in PBS for 30 min and 30% sucrose in PBS for 30 min. Retinas were embedded in cryomatrix (Tissuetek, Bayer Corporation, Pittsburgh, PA, USA), frozen and stored at –80°C. Radial sections of 7 µm were cut on a cryostat, collected onto Poly-L-lysine (Sigma-Aldrich, p1274, St Louis, MO, USA) coated object glasses, air-dried and stored at –20°C or at –80°C for further processing.


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  		Table 1. Number of retinas used

 
Immunofluorescence microscopy
Paraffin-embedded sections (4 µm) or cryosections (7 µm) of WT or Crb1–/– cultured retinas (P1.5-DIV17) were incubated over night with various primary antibodies: monoclonal antibody (mAb) against E-cadherin, N-cadherin, Mupp1, p120, N-cadherin and ß-catenin (Becton Dickinson Transduction laboratories, Austria); actin (Mab1501R, Chemicon, Temecula, CA, USA) and polyclonal antibody (pAb) directed against p34-Arc (Upstate Biotechnologies Inc., Lake Placid, NY, USA); Veli3, GFP (Zymed, San Francisco, USA); CD-44 (kindly provided by R. van der Neut); rhodopsin (kindly provided by W.J. de Grip) and ZO-1 and Glutamate Synthetase (Becton Dickinson). Rabbit polyclonal antibody against Patj (5–8) and CRB3 (12) were described previously. Polyclonal antibody against Mupp1 was kindly provided by Dr A. Le Bivic (CNRS, Marseille, France). Epitopes used to raise antibodies against Crb1, Crb2, Mpp4 and Pals1 were described earlier (3,41,48). Fluorescent-labelled secondary antibodies were donkey anti-chicken, goat anti-mouse or goat anti-rabbit IgGs conjugated to Cy3, Alexa488 or FITC (Jackson Immuno Research, Stanford, USA, and Invitrogen). Immunofluoresence stainings were performed as previously described (47). The sections were visualized by confocal laser scanning microscopy (Zeiss LSM510, Jena, Germany) and pictures were taken using Zeiss LSM browser v3.2 image software.

Immuno-EM
Pre-embedding.
For pre-embedding immunohistochemistry (37) of Crb1, two WT and two Crb1–/– male animals at the age of 3 months were perfused for 1 min with 0.1 M sodium cacodylate in PBS, pH 7.4 and 5 min with 4% PF in phosphate buffer 0.1 M, pH 7.4. Eyes were enucleated and fixed in 4% PF in PBS for 30 min followed by cryo-protection with sucrose (5 and 30%) and stored at –80°C. Frozen sections of 40 µm thick were cut on the cryostat and collected in phosphate buffer. Sections were incubated for 96 h with primary antibody (anti-Crb1 AK2, 1:200) (3) (anti-Pals1 SN47, 1:100). After rinsing with PB, the sections were incubated with secondary antibody ImmunoVision Poly-HRP-Goat Anti-rabbit IgG (ImmunoVision Technologies Co., Daly City, CA, USA). A Tris–HCl diaminobenzidine (DAB) (Sigma-Aldrich) solution containing 0.03% H2O2 (Sigma) was used to visualize the peroxidase present on the secondary antibody and intensified by the gold-substituted silver peroxidase method (50). Sections were rinsed in sodium cacodylate buffer (0.1 M, pH 7.4) and post-fixed for 20 min in 1% osmium supplemented with 1% potassium ferricyanide in sodium cacodylate buffer (0.1 M, pH 7.4). After a second wash with sodium cacodylate buffer, the material was dehydrated and embedded in epoxy resin as described above. Ultrathin sections were cut from both immunostained sections and photographed using the FEI TECHNAI electron microscope. Pictures were collected with the ImageView soft imaging system and processed using Adobe photoshop.

Post-embedding.
For the morphological study, adult mice were perfused with 4% PF+2% glutaraldehyde in cacodylate buffer pH 7.4. After the retinas were dissected free, they were post-fixed for 1 h in 1% osmium tetroxide in the same buffer. Small specimens were then prepared and processed as previously described (51). For immunoelectron localization of Crb2, Crb3, Mupp1 and Patj, adult mice were perfused with 4% PFA in PBS and removed retinas were prepared and processed for immunolabelling as recently described for Caco-2 cells (38). The cryosubstitution method used in this study is a slight modification of that previously described for mammalian retina (52). The ultrathin sections were examined with a Zeis 912 electron microscope (Zeiss, Le Pecq. France).

Plasmid constructs
For Pals1 silencing by RNAi, a DNA construct was generated by annealing DNA oligonucleotides 5'-agcttttccaaaaaGTTCATTGAACATGGTGAAtctcttgaaTTCACCATGTTCAATGAACggg-3' and 5'-gatccccGTTCATTGAACATGGTGAAttcaagagaTTCACCATGTTCAATGAACtttttggaaa-3', corresponding to nucleotides 1970–1988 of the GUK domain of mouse Pals1 (3-shPals1). For Mpp4 silencing, DNA oligonucleotides 5'-agcttttccaaaaaGGATGAAGACCTACAAGAGAtctcttgaaCTCTTGTAGGTCTTCATCCggg-3' and 5'-gatccccGGATGAAGACCTACAAGAGAttcaagagaCTCTTGTAGGTCTTCATCCtttttggaaa-3' corresponding to nucleotides 1726–1744 of the GUK domain of mouse Mpp4 were annealed. The primers were purchased from Eurogentec (Seraing, Belgium). Annealed primers were cloned into the HindIII and BglII sites of the pSUPER vector (40). The empty pSUPER vector was used as a control. pSUPER–Pals1 or pSUPER–Mpp4 RNAi constructs were co-transfected in HEK293T cells with GFP-tagged full-length Mpp4 or GFP-tagged Pals1 oligos (5'-gatctcAAGTTCATTGAACATGGTGAATTgca-3' annealed to 5'-agcttgcAATTCACCATGTTCAATGAACTTa-3' and subcloned into HindIII/BglII of pEGFP-C1). Five days after transient transfection, cells were lysed and protein was evaluated by immunoblot analysis with anti-GFP. The pSUPER–Pals1 (3-shPals1) and pSUPER–Mpp4 vectors were digested with XbaI–KpnI and blunted. The shPals1 and shMpp4 fragments including the H1 promotor were blunt ligated into an AAV2–CMV-GFP vector (53) which was digested with SphI (two sites after the WPRE and polyA) and blunted. The CMV-promoter-driven hCRB1 construct was generated by introducing hCRB1 cDNA (SpeI/blunt and XhoI) into AAV2–CMV vector (ApaI/blunt and XhoI).

Western blotting
Cells were washed in ice-cold PBS, lysed in 400 µl lysis buffer [50 mM HEPES, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 1x Complete protease inhibitors-EDTA (Roche, Woerden, The Netherlands), 10 µg/ml aprotinin (Sigma), pH 7.4] and were tumbled at 4°C for 45 min. Cells extracts were pre-cleared by 15 min centrifugation and protein concentration was determined using Biorad Protein assay kit (Pierce, Rockford, USA). Lysates were boiled in Laemmli sample buffer, containing ß-mercapto-ethanol and separated by 9% SDS–PAGE. Proteins were transferred onto nitrocellulose membranes and stained with Ponceau S. Membranes were blocked [1% milk, 1% BSA in Tris-buffered saline with Tween-20 (TBST; 50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween-20)]. Protein detection was performed by probing the membranes with primary antibodies against Pals1 or Mpp4 in 1:300 dilution in blocking buffer for at least 1 h, washed three times in TBST, incubated with 1:10 000 dilution of horseradish peroxidase-conjugated secondary anti-mouse and anti-rabbit antibody (Jackson Immuno Research en Bio-rad Laboratories, Hercules, CA, USA) in blocking buffer for 1 h. Proteins were visualized using chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL, USA).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
References
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Reuven Agami for providing the pSUPER-vector, Joost Verhaagen for providing the rAAV2-CMV-GFP vector, R. van der Neut for anti-CD44, W.J. de Grip for anti-rhodopsin, Bob Nunes Cardozo for technical assistance with confocal and electron microscopy, Gerben van der Meulen for photographic assistance, Romeo Caffé for technical advice for the retinal cultures, the members of the animal facility of the Netherlands Institute for Neuroscience for animal care and Serge A. van de Pavert and Albena Kantardzhieva for helpful discussions. This work was supported in part by grant 912-02-018 from ZonMW-NWO (to J.W.), Stichting OOG and Rotterdamse Vereniging Blindenbelangen (to J.W.), grant QLG3-CT-2002-01266 from the European Commission (EC) (to J.W.), Van Gogh travel grants (to J.W. and A.L.B.) and by a grant from Agence Nationale pour la Recherche, Neuroscience program (to A.L.B.).

Conflict of Interest statement. None declared.


   References
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 

  1. Malicki J. (2004) Cell fate decisions and patterning in the vertebrate retina: the importance of timing, asymmetry, polarity and waves. Curr. Opin. Neurobiol. 14:15–21.[CrossRef][Web of Science][Medline]

  2. Masai I., Lele Z., Yamaguchi M., Komori A., Nakata A., Nishiwaki Y., Wada H., Tanaka H., Nojima Y., Hammerschmidt M., et al. (2003) N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites. Development 130:2479–2494.[Abstract/Free Full Text]

  3. van de Pavert S.A., Kantardzhieva A., Malysheva A., Meuleman J., Versteeg I., Levelt C., Klooster J., Geiger S., Seeliger M.W., Rashbass P., et al. (2004) Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure. J. Cell Sci. 117:4169–4177.[Abstract/Free Full Text]

  4. Kamberov E., Makarova O., Roh M., Liu A., Karnak D., Straight S., Margolis B. (2000) Molecular cloning and characterization of Pals, proteins associated with mLin-7. J. Biol. Chem. 275:11425–11431.[Abstract/Free Full Text]

  5. Makarova O., Roh M.H., Liu C.J., Laurinec S., Margolis B. (2003) Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Pals1). Gene 302:21–29.[CrossRef][Web of Science][Medline]

  6. Roh M.H., Makarova O., Liu C.J., Shin K., Lee S., Laurinec S., Goyal M., Wiggins R., Margolis B. (2002) The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol. 157:161–172.[Abstract/Free Full Text]

  7. Lemmers C., Medina E., Delgrossi M.H., Michel D., Arsanto J.P., Le Bivic A. (2002) hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J. Biol. Chem. 277:25408–25415.[Abstract/Free Full Text]

  8. Li Y., Karnak D., Demeler B., Margolis B., Lavie A. (2004) Structural basis for L27 domain-mediated assembly of signaling and cell polarity complexes. EMBO J. 23:2723–2733.[CrossRef][Web of Science][Medline]

  9. Ohno S. (2001) Intercellular junctions and cellular polarity: the PAR–aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr. Opin. Cell Biol. 13:641–648.[CrossRef][Web of Science][Medline]

  10. Suzuki A., Yamanaka T., Hirose T., Manabe N., Mizuno K., Shimizu M., Akimoto K., Izumi Y., Ohnishi T., Ohno S. (2001) Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J. Cell Biol. 152:1183–1196.[Abstract/Free Full Text]

  11. Tabuse Y., Izumi Y., Piano F., Kemphues K.J., Miwa J., Ohno S. (1998) Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans. Development 125:3607–3614.[Abstract]

  12. Lemmers C., Michel D., Lane-Guermonprez L., Delgrossi M.H., Medina E., Arsanto J.P., Le Bivic A. (2004) CRB3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells. Mol. Biol. Cell 15:1324–1333.[Abstract/Free Full Text]

  13. Koike C., Nishida A., Akimoto K., Nakaya M.A., Noda T., Ohno S., Furukawa T. (2005) Function of atypical protein kinase Clambda in differentiating photoreceptors is required for proper lamination of mouse retina. J. Neurosci. 25:10290–10298.[Abstract/Free Full Text]

  14. Wei X., Cheng Y., Luo Y., Shi X., Nelson S., Hyde D.R. (2004) The zebrafish Pard3 ortholog is required for separation of the eye fields and retinal lamination. Dev. Biol. 269:286–301.[CrossRef][Web of Science][Medline]

  15. Bachmann A., Schneider M., Theilenberg E., Grawe F., Knust E. (2001) Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature 414:638–643.[CrossRef][Web of Science][Medline]

  16. Hong Y., Ackerman L., Jan L.Y., Jan Y.N. (2003) Distinct roles of Bazooka and Stardust in the specification of Drosophila photoreceptor membrane architecture. Proc. Natl Acad. Sci. USA 100:12712–12717.[Abstract/Free Full Text]

  17. Bhat M.A., Izaddoost S., Lu Y., Cho K.O., Choi K.W., Bellen H.J. (1999) Discs lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity. Cell 96:833–845.[CrossRef][Web of Science][Medline]

  18. Muller H.A. and Wieschaus E. (1996) armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134:149–163.[Abstract/Free Full Text]

  19. Tepass U., Tanentzapf G., Ward R., Fehon R. (2001) Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35:747–784.[CrossRef][Web of Science][Medline]

  20. Richard M., Grawe F., Knust E. (2005) DPATJ plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye. Dev. Dyn. 235:895–907.

  21. Nam S.C. and Choi K.W. (2006) Domain-specific early and late function of Dpatj in Drosophila photoreceptor cells. Dev. Dyn. 235:1501–1507.[CrossRef][Web of Science][Medline]

  22. Izaddoost S., Nam S.C., Bhat M.A., Bellen H.J., Choi K.W. (2002) Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416:178–183.[CrossRef][Medline]

  23. Pellikka M., Tanentzapf G., Pinto M., Smith C., McGlade C.J., Ready D.F., Tepass U. (2002) Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature 416:143–149.[CrossRef][Medline]

  24. Johnson K., Grawe F., Grzeschik N., Knust E. (2002) Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration. Curr. Biol. 12:1675–1680.[CrossRef][Web of Science][Medline]

  25. den Hollander A.I., ten Brink J.B., de Kok Y.J., van Soest S., van den Born L.I., van Driel M.A., van de Pol D.J., Payne A.M., Bhattacharya S.S., Kellner U., et al. (1999) Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12). Nat. Genet. 23:217–221.[CrossRef][Web of Science][Medline]

  26. den Hollander A.I., Heckenlively J.R., van den Born L.I., de Kok Y.J., van der Velde-Visser S.D., Kellner U., Jurklies B., van Schooneveld M.J., Blankenagel A., Rohrschneider K., et al. (2001) Leber congenital amaurosis and retinitis pigmentosa with coats-like exudative vasculopathy are associated with mutations in the crumbs homologue 1 (CRB1) gene. Am. J. Hum. Genet. 69:198–203.[CrossRef][Web of Science][Medline]

  27. den Hollander A.I., Davis J., van der Velde-Visser S.D., Zonneveld M.N., Pierrottet C.O., Koenekoop R.K., Kellner U., van den Born L.I., Heckenlively J.R., Hoyng C.B., et al. (2004) CRB1 mutation spectrum in inherited retinal dystrophies. Hum. Mutat. 24:355–369.[CrossRef][Web of Science][Medline]

  28. McKay G.J., Clarke S., Davis J.A., Simpson D.A.C., Silvestri G. (2005) Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene. Invest. Ophthalmol. Vis. Sci. 46:322–328.[Abstract/Free Full Text]

  29. Mehalow A.K., Kameya S., Smith R.S., Hawes N.L., Denegre J.M., Young J.A., Bechtold L., Haider N.B., Tepass U., Heckenlively J.R., et al. (2003) CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Hum. Mol. Genet. 12:2179–2189.[Abstract/Free Full Text]

  30. Roh M.H., Fan S., Liu C.J., Margolis B. (2003) The Crumbs3–Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J. Cell Sci. 116:2895–2906.[Abstract/Free Full Text]

  31. Straight S.W., Shin K., Fogg V.C., Fan S., Liu C.J., Roh M., Margolis B. (2004) Loss of PALS1 expression leads to tight junction and polarity defects. Mol. Biol. Cell 15:1981–1990.[Abstract/Free Full Text]

  32. Wei X. and Malicki J. (2002) Nagie oko, encoding a MAGUK-family protein, is essential for cellular patterning of the retina. Nat. Genet. 31:150–157.[CrossRef][Web of Science][Medline]

  33. Wei X., Zou J., Takechi M., Kawamura S., Li L. (2006) Nok plays an essential role in maintaining the integrity of the outer nuclear layer in the zebrafish retina. Exp. Eye Res. 83:31–44.[CrossRef][Web of Science][Medline]

  34. Caffe A.R., Visser H., Jansen H.G., Sanyal S. (1989) Histotypic differentiation of neonatal mouse retina in organ culture. Curr. Eye Res. 8:1083–1092.[Web of Science][Medline]

  35. Ogilvie J.M., Speck J.D., Lett J.M., Fleming T.T. (1999) A reliable method for organ culture of neonatal mouse retina with long-term survival. J. Neurosci. Methods 87:57–65.[CrossRef][Web of Science][Medline]

  36. Chen H. and Weber A.J. (2002) Expression of glial fibrillary acidic protein and glutamine synthetase by Muller cells after optic nerve damage and intravitreal application of brain-derived neurotrophic factor. Glia 38:115–125.[CrossRef][Web of Science][Medline]

  37. Klooster J., Nunes C.B., Yazulla S., Kamermans M. (2004) Postsynaptic localization of gamma-aminobutyric acid transporters and receptors in the outer plexiform layer of the goldfish retina: An ultrastructural study. J. Comp. Neurol. 474:58–74.[CrossRef][Web of Science][Medline]

  38. Michel D., Arsanto J.P., Massey-Harroche D., Beclin C., Wijnholds J., Le Bivic A. (2005) PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells. J. Cell Sci. 118:4049–4057.[Abstract/Free Full Text]

  39. Matsuda T. and Cepko C.L. (2004) Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl Acad. Sci. USA 101:16–22.[Abstract/Free Full Text]

  40. Brummelkamp T.R., Bernards R., Agami R. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2:243–247.[CrossRef][Web of Science][Medline]

  41. Kantardzhieva A., Gosens I., Alexeeva S., Punte I.M., Versteeg I., Krieger E., Neefjes-Mol C.A., den Hollander A.I., Letteboer S.J., Klooster J., et al. (2005) MPP5 recruits MPP4 to the CRB1 complex in photoreceptors. Invest. Ophthalmol. Vis. Sci. 46:2192–2201.[Abstract/Free Full Text]

  42. den Hollander A.I., Ghiani M., de Kok Y.J., Wijnholds J., Ballabio A., Cremers F.P., Broccoli V. (2002) Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression pattern in eye and brain. Mech. Dev. 110:203–207.[CrossRef][Web of Science][Medline]

  43. Watanabe T., Miyatani S., Katoh I., Kobayashi S., Ikawa Y. (2004) Expression of a novel secretory form (Crb1s) of mouse crumbs homologue Crb1 in skin development. Biochem. Biophys. Res. Commun. 313:263–270.[CrossRef][Web of Science][Medline]

  44. Harada T., Harada C., Kohsaka S., Wada E., Yoshida K., Ohno S., Mamada H., Tanaka K., Parada L.F., Wada K. (2002) Microglia–Muller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration. J. Neurosci. 22:9228–9236.[Abstract/Free Full Text]

  45. Lewis G.P. and Fisher S.K. (2000) Muller cell outgrowth after retinal detachment: association with cone photoreceptors. Invest. Ophthalmol. Vis. Sci. 41:1542–1545.[Abstract/Free Full Text]

  46. Stohr H., Molday L.L., Molday R.S., Weber B.H., Biedermann B., Reichenbach A., Kramer F. (2005) Membrane-associated guanylate kinase proteins MPP4 and MPP5 associate with Veli3 at distinct intercellular junctions of the neurosensory retina. J. Comp. Neurol. 481:31–41.[CrossRef][Web of Science][Medline]

  47. Aartsen W.M., Kantardzhieva A., Klooster J., van Rossum A.G., van de Pavert S.A., Versteeg I., Cardozo B.N., Tonagel F., Beck S.C., Tanimoto N., et al. (2006) Mpp4 recruits Psd95 and Veli3 towards the photoreceptor synapse. Hum. Mol. Genet. 15:1291–1302.[Abstract/Free Full Text]

  48. Kantardzhieva A., Alexeeva S., Versteeg I., Wijnholds J. (2006) Mpp3 is recruited to the Mpp5 protein scaffold at the retinal outer limiting membrane. FEBS J. 273:1152–1165.[CrossRef][Medline]

  49. Roh M.H., Liu C.J., Laurinec S., Margolis B. (2002) The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions. J. Biol. Chem. 277:27501–27509.[Abstract/Free Full Text]

  50. van den Pol A.N. and Gorcs T. (1986) Synaptic relationships between neurons containing vasopressin, gastrin-releasing peptide, vasoactive intestinal polypeptide, and glutamate decarboxylase immunoreactivity in the suprachiasmatic nucleus: dual ultrastructural immunocytochemistry with gold-substituted silver peroxidase. J. Comp. Neurol. 252:507–521.[CrossRef][Web of Science][Medline]

  51. Breuza L., Corby S., Arsanto J.P., Delgrossi M.H., Scheiffele P., Le Bivic A. (2002) The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells. J. Cell Sci. 115:4457–4467.[Abstract/Free Full Text]

  52. Brandstatter J.H., Fletcher E.L., Garner C.C., Gundelfinger E.D., Wassle H. (1999) Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina. Eur. J. Neurosci. 11:3683–3693.[CrossRef][Web of Science][Medline]

  53. Blits B. Cell and gene therapy and experimental strategies for spinal cord regeneration. Thesis/Dissertation,, Vrije Universiteit Amsterdam.

  54. Pinzon-Duarte G., Kohler K., Arango-Gonzalez B., Guenther E. (2000) Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture. Vision Res. 40:3455–3465.[CrossRef][Web of Science][Medline]


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